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Autophagy-related gene 5 (Atg5) plays an essential role in autophagy, the loss of its function impairs neurogenesis and axon regeneration. However, the biological function of Atg5 has not been characterized in planarian. Planarian is an ideal model for the study of brain regeneration. It can regenerate a new brain de novo in 1 week following amputation. To explore the role of Atg5 in planarian brain regeneration, we dissected the molecular characteristics of Atg5 in planarian Dugesia japonica (DjAtg5) and examined its function by RNAi. The full-length cDNA of DjAtg5 is 1 014 bp encoding 284 amino acids. The deduced amino sequence of DjAtg5 contains the functional Pfam domain of ATG5 and highly conserved residues for ATG5-ATG12 interaction. After amputation, the transcrips of DjAtg5 are increased and mainly distributed in the newly regenerated brain on day 3-5 of regeneration. However, knockdown of DjAtg5 by RNAi does not impair the regeneration ability and brain structure reformation, nor affects the neoblasts proliferation. Our results suggest that DjAtg5 participates in re-formation of planarian brain structure following amputation, but it is not an important regulator for planarian regeneration. However, autophagy inhibitor 3-MA can block planarian regeneration, which suggests that autophagy is necessary for planarian regeneration.
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Farnesol (FOH) is produced by dephosphorylation of farnesyl diphosphate (FPP) derived from two universal building blocks, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In Rhodobacter sphaeroides these building blocks are generated by MEP pathway, however, many of the biosynthetic reactions and biotransformations in the MEP pathway are limited by low availability of NADPH. Improvement of the amount of intracellular NADPH may enhance the synthesis of FOH. In this study, we utilized the strategies of increasing the production of NADPH and decreasing the consumption of NADPH. The expression of glucose 6-phosphate isomerase (pgi) and glutamate dehydrogenase (gdhA) were inhibited by RNA interference, respectively, and overexpression of 6-glucose phosphate dehydrogenase (zwf) and 6-glucose phosphate dehydrogenase (gnd) in the pentose phosphate pathway were carried out. The results showed that the content of NADPH in the recombinant strains increased significantly, the highest FOH production of RSpgii in the RNA interfered strain was 3.91 mg/g, and the FOH production increased to 3.43 mg/g after zwf gene and gnd gene has been overexpressed. In order to obtain strains with higher FOH production, we used RSpgii as the starting strain, and zwf, gnd and co-overexpressed zwf + gnd gene were overexpressed in RSpgii, respectively. The highest FOH production of the strain RSzgpi reached to 4.48 mg/g which was 2.24 times that of the starting strain RS-GY2.
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Objective: To investigate the effects of DNAJ homolog subfamily B member 11 (DNAJB 11) gene-silencing on proliferation, cell cycle and apoptosis of human hepatocellular carcinoma cell line SMMC7721. Methods: The recombinant lentiviral vector pCDH-Puro/DNAJB11-shRNA carrying the specific shRNA targeting DNAJB 11 gene was established. The SMMC7721 cells were infected with high infective lentivirus pCDH-Puro/DNAJB11-shRNA. Then the proliferation of SMMC7721 cells was detected by CCK-8 method. The expression levels of DNAJB11, proliferating cell nuclear antigen (PCNA) and caspase-3 mRNAs and proteins in SMMC7721 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The cell cycle distribution and the apoptosis rate of SMMC7721 cells were analyzed by FCM. Results: The pCDH-Puro/DNAJB11-shRNA was constructed successfully. The proliferation of SMMC7721 cells was significantly inhibited after infection with pCDH-Puro/DNAJB11- shRNA (P<0.05). In SMMC7721 cells infected with pCDH-Puro/DNAJB11-shRNA, the expressions of DNAJB11 mRNA and protein were silenced effectively (both P<0.05). After DNAJB 11 gene-silencing, the expressions of caspase-3 mRNA and protein in SMMC7721 cells were up-regulated (both P<0.05), while the expressions of PCNA mRNA and protein were down-regulated (both P<0.05). Furthermore, the cell cycle was arrested in G1 phase (P<0.01), and the apoptosis rate was significantly increased (P<0.01). Conclusion: The DNAJB 11 gene-silencing can effectively suppress the proliferation of SMMC7721 cells, and promote their apoptosis. These effects may be related to downregulation of PCNA expression and upregulation of caspase-3 expression in SMMC7721 cells.
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Background Age-related macular degeneration (AMD) is the leading cause of blindness in people over 50 years old,of which 90％ cases are caused by choroidal neovascularization (CNV).Current treatments on AMD have gained great achievements,but there are still some drawbacks.So we need to search for new targets to cure CNV.Objective This study was to construct two combined lentiviral vectors for rat Slit2 gene RNA interference (RNAi) and verify its interfering effects on Slit2 gene in rat retinal pigment epithelial (RPE) cells.Methods Two specific siRNA sequences targeting towards rat Slit2 gene were designed and were annealed to DNA sequences.The DNA sequences and GV248-enhanced green flourescent protein (EGFP) vectors were combined together as recombinant vectors and then were identified.The GV248-EGFP vector,helper 1.0 and helper 2.0 were transfected together into 293T cells and the two combined lentiviral vectors for rat Slit2 RNAi were gained from the cell supernatant after 72 hours of transfection.The titers of the combined lentiviral vectors were measured.The cells were divided into blank control group,Lv-EGFP vector group,Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group.The interference efficacy of the combined lentiviral vectors targeting to rat Slit2 gene were identified by real-time fluorescence quantitative PCR and Western blot.The sequence with higher interference efficacy was transfected to rat RPE cells again.The transfected and nontransfected rat RPE cells were treated with 0,100,200 and 400 μmol/L CoCl2 for the preparation of hypoxia models.The expression of vascular endothelial growth factor-A (VEGFA) mRNA in rat RPE cells was finally measured by real-time fluorescence quantitative PCR and the concentration of VEGFA protein in cell supernatant was assayed by ELISA.Results The recombined lentiviral vectors for rat Slit2 gene R NAi were successfully constructed.The titers of the two reconbinant sequences were 5× 108 TU/ml and 3×l08 TU/ml,with the transfected rate ≥70％.The relative expression levels of Slit2 mRNA in the Lv-rSlit2-siRNA1 group and LvrSlit2-siRNA2 group were 0.67±0.09 and 0.23±0.11,respectively,which were lower than 1.03±0.31 and 0.92± 0.07 in the blank control group and Lv-EGFP vector group (all at P＜0.01).The expression levels of Slit2 protein in the Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group were 0.62±0.07 and 0.49±0.02,respectively,which were lower than 1.00±0.10 in blank control group and 0.95±0.11 in Lv-EGFP vector group (all at P＜0.01).Significant differences were found in the expression of VEGFA mRNA and protein in RPE cells among different concentrations of CoC12 groups (mRNA:F tration =127.998,P＜0.01;Fgroup =69.663,P＜0.01.Protein:F ion =17.059,P＜ 0.01;Fgroup =91.791,P＜0.01),and the expression of VEGFA mRNA and the concentration of VEGFA protein were evidently lower in the Lv-rSlit2-siRNA2 group than those in the blank control group after being treated by 100,200 and 400 μmol/L CoCl2 (all at P＜0.01).Conclusions Recombined lentiviral vector for rat Slit2 gene RNAi is successfully constructed,which can effectively knockdown rat Slit2 gene and inhibit the expression of VEGFA in rat RPE cells.
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Objective To investigate the effects of c?myc promoter binding protein 1(MBP?1)gene on the proliferation of human Saos?2 osteo?sarcoma cells in vitro. Methods Saos?2 cells were divided into three groups:blank control group(untransfected cells),negative group(cells transfected with missense sequence)and experimental group(cells transfected with MBP?1 shRNA). Two MBP?1 shRNA sequences and one neg?ative control shRNA sequence were designed ,synthesized and cloned into pSIREN?retroQ plasma. Then the recombinant plasmids were construct?ed and transfected into human Saos?2 osteosarcoma cells by Lipofectamine 2000. The expressions of MBP?1 mRNA and protein in Saos?2 cells were detected by real?time PCR and Western blot ,respectively. The effects of altered expression of MBP?1 on cell proliferation were measured by CCK?8 cell proliferation assay. The expressions of cyclin D1 and cyclin E in Saos?2 were determined by Western blot. Results PCR and sequenc?ing results indicated that the recombinant plasmids pSIREN?retroQ was constructed. The relative expression level of MBP?1 mRNA in the MBP?1 siRNA transfection group was significantly decreased than that in blank control group(P<0.05). Compared with the blank control group,the ex?pression levels of MBP?1 protein in the experimental group also significantly decreased. The proliferation abilities of Saos?2 cells at 48,72,and 96 hours after MBP?1 siRNA transfection were significantly increased than those in the blank control group(P<0.05). Compared with the blank con?trol group,the expression levels of cyclin D1 and cyclin E protein in the experimental group also significantly increased(P<0.05). Conclusion Knockdown of the expression of MBP?1 gene promotes the proliferation of human Saos?2 osteosarcoma cells. MBP?1 gene may become the new tar?get of gene therapy for osteosarcoma.
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Objective To investigate the effects of c-myc promoter binding protein(MBP-1)gene expression silencing on the pro-liferation in vitro in human gastric cancer cell line SGC-7901.Methods The cells divided into three groups:blank control group (cells without transfecting gastric cancer cell),negative control group(cells transfecting missense sequence)and experimental group (cells transfecting MBP-1 shRNA).Two MBP-1 shRNA sequences and one negative control shRNA sequence were designed,syn-thesized and cloned into pSIREN-retroQ plasma.Then the recombinant plasmids were constructed and transfected into human gas-tric cancer SGC-7901 cells by Lipofectamine 2000.After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was obtained.The expressions of MBP-1 mRNA and protein in SGC-7901 were deter-mined by the real time PCR and Western blot,respectively.The effects of altered expression of MBP-1 on the cell proliferation were measured by MTT cell proliferation assay.Results PCR and sequencing indicated that the recombinant plasmids pSIREN-retroQ was constructed.Then the recombinant plasmids were transfected into human gastric cancer SGC-7901 cells by Lipofectamine 2000. After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was ob-tained.The relative expression level MBP-1 mRNA in the MBP-1 siRNA transfection group was significantly decreased compared with the blank control group(P <0.05).Compared with the blank group,the expression levels of MBP-1 protein in the experimental group also significantly decreased.The proliferation abilities of SGC-7901 cells at 48,72,96,120 h after MBP-1 siRNA transfection were significantly increased compared with the blank control group (P < 0.05 ).Conclusion Down-regulating the expression of MBP-1 can obviously promote the proliferation of human gastric cancer cell line SGC-7901.MBP-1 gene may become the new target of gene therapy for gastric cancer.
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OBJECTIVE@#To observe the clinical manifestations of allergic rhinitis mice and the expression changes of the eosinophils CCR3 and the granule protein mRNA in the bone marrow, peripheral blood and nasal lavage fluid.@*METHODS@#Twenty-four BALB/c mice were randomly divided into the control group, PBS therapy group, siRNA therapy group and the CCR3 siRNA therapy group (n=6). Allergic rhinitis model were sensitized and stimulated by ovalbunfin, and CCR3 siRNA therapy group were administered with CCR3 transnasally before stimulated. The levels of the eosinophils CCR3, MBP, ECP and EPO in bone marrow, peripheral blood and nasal lavage fluid were detected by RT-PCR.@*RESULTS@#Compared to the control group and CCR3 siRNA therapy group, the nasal mucosa of the PBS therapy group and siRNA therapy group developed epithalaxy, goblet cells hyperplasia, squamous epithelium metaplasia, epithelium necrosis, lamina propria and submucosa gland hyperplasia, vasodilatation, tissue edema, and the characterized eosinophil infiltration. RT-PCR indicated that the CCR3 mRNA, MBP, ECP and EPO expression in bone marrow, peripheral blood and nasal lavage fluid of the CCR3 siRNA therapy group was lower than the PBS therapy group and siRNA therapy group (P<0.05).@*CONCLUSIONS@#The RNA interference therapy to CCR3 by local administration pernasal can suppress the process of the development, migration and invasion of the allergic rhinitis eosinophil, thus can reduce the effect of eosinophils and then reduce the inflammation effect of the allergic rhinitis. It may be a new treatment for respiratory tract allergic inflammation.
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Animales , Masculino , Ratones , Conducta Animal , Médula Ósea , Química , Modelos Animales de Enfermedad , Proteínas en los Gránulos del Eosinófilo , Genética , Metabolismo , Eosinófilos , Metabolismo , Fisiología , Ratones Endogámicos BALB C , Mucosa Nasal , Química , Biología Celular , ARN Interferente Pequeño , Genética , Distribución Aleatoria , Receptores CCR3 , Genética , Metabolismo , Rinitis Alérgica , Rinitis Alérgica Perenne , Genética , TerapéuticaRESUMEN
Objective To explore the influence of EGFR gene interfering on the radiosensitivity of esophageal squamous cells.Methods Three kinds of siRNAs including three random sequences of positive EGFR siRNA (EGFR siRNA1、EGFR siRNA2、EGFR siRNA3),random sequence of negative EGFR siRNA,and blank control were transfected into Eca109 cells by lipofectamine.Cell proliferation was detected by Cell Counting Kit-8 assay.Protein and mRNA expressions of EGFR were detected by Western blot and RT-PCR,respectively.The ability of cell clone formation was used to evaluate the combination effect of X-rays and EGFR siRNA on the radiosensitivity.Results The positive expression rate of the EGFR mRNA in the Eca109 ceils transfected with EGFR siRNA1,EGFR siRNA2,EGFR siRNA3 was 26.74％,9.52％,4.61％,respectively,which was significantly lower than 42.44％ in the control cells transfected with blank siRNA (F =112.11,P ＜ 0.01).Meanwhile,the EGFR protein expression was reduced by 72.84％,53.01％ and 56.21％ after interfering of siRNA1,siRNA2,and siRNA3,respectively.CCK8 assay showed that the proliferation of Eca109 cells was decreased by 28.2％ since the siRNA interference.Moreover,the D0,Dq and SF2 of the combined treatment group were lower than those of irradiation alone group and the sensitization enhancement ratio was 1.50.Conclusions EGFR siRNA can effectively inhibit EGFR gene expression and enhance the radiosensitivity of Eca109 cells.
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Objective To investigate the specific silencing of connective tissue growth factor (CTGF) in a nude mouse keloid model,using RNA interference (RNAi) technique,and to provide the basis for gene therapy of keloid.Methods The nude mouse keloid model was established,and then transfected in vivo with well-amplifiating plasmid.The mRNA expression levels of CTGF mRNA and type Ⅰ collagen mRNA were detected with reverse transcription-polymerase chain reaction (RT-PCR).The distribution and protein expression levels of CTGF and type Ⅰ collagen were determined quantitatively using immunohistochemistry.Results The expression of CTGF at mRNA and protein levels was decreased in the experiment group,and the expression of type Ⅰ collagen at mRNA and protein levels was also decreased after transfection,as compared with negative control group and blank group,with significant difference between groups (P＜0.05).Moreover,the expression of type Ⅰ collagen and CTGF was positively correlated (r=0.979).Conclusions Keloid type Ⅰ collagen can be decreased through in vivo inhibiting CTGF expression.The transfection of CTGF gene in vivo may have effects on type Ⅰ collagen generation,and thus inhibit the keloid growth.
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ObjectiveTo investigate the in vivo effects of down-regulating the FGF-21 gene expression by shRNA on glucose and lipids metabolism in high fat diet (HFD) fed ApoE-/- mice.MethoedsMale ApoE-/- mice were randomly divided into chow diet (CF)fed group ( NF,n =10),CF fed + pAd-shFGF-21 group ( NFG,n =9),and HFD fed group ( HF,n =10),HFD fed + Adv-null vector ( pAd-GFP ) group ( GFP,n =6) and HFD fed + pAd-shFGF-21 group ( HFG,n =10).Mice were fed for 16 weeks.C57BL/6J mice were set as control group ( NC group,n=10).NFG,HFG,and GFP groups were injected with pAd-shFGF-21or pAd-GFP by tail vein at the end of 15 weeks.The insulin sensitivity and glucoselipid metabolism were assessed by the hyperinsulinemic- euglycemic clamp technique using 3-[ 3 H] glucose as a tracer at the end of 16 week.ResultsThe plasma FGF-21 levels in NFG and HFG groups were significantly degraded than those in NF and HF groups(20％-27％,P＜0.05),respectively.In the basal state,the fasting blood glucose,fasting plasma insulin,free fatty-acids,triglycerides,total cholesterol,and LDL-C were significantly higher,while the HDL-C was lower in NFG and HFG groups compared with those in NF and HF groups,respectively (P＜0.05 or P＜0.01 ).During the steady-state of clamp,FFA was suppressed in all groups,but it was still higher in NFG and HFG groups than NF and HF groups ( P＜0.05or P＜0.01 ).The glucose infusion rate (GIR)and glucose disappearance rate (GRd)in NFG and HFG groups were significantly decreased compared with NF and HF groups (all P＜0.01 ).In addition,insulin's ability to suppress hepatic glucose production (HGP) during clamps was significantly decreased in HFG and NFG group compared with HF and NF groups (49％ and 20％,respectively; all P＜0.01 ).ConclusionFGF-21 knockdown and low FGF-21 level facilitate the development of metabolic disorder and insulin resistance.
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Objective:To establish the RPB5-mediating protein (RMP)-silenced stable cell lines and study the inhibitory effects of small interfering RNA (siRNA) targeting RMP gene on the proliferation and migration of human hepatoma SMMC-7721 cells. Methods:Three RMPi siRNAs were designed and synthesized in vitro and transfected into SMMC-7721 cells. The inhibitory effect of siRNA on RMP gene expression was measured by RT-PCR to select the best siRNA. The expression vector pGPU6-Neo-RMP-484 was transfected into SMMC-7721 cells by the lipofectamine and the cells stably expressing the siRNA were selected by G418. RT-PCR was used to detect the interference efficacy against RMP gene. Cell proliferation and adhesion were measured by MTT assay. Wound healing test was used to observe the migration ability of cells. Results:The SMMC-7721 cell lines with down-regulated RMP expression were established by using RNA interference technology. Compared with the negative control cells, expression of RMP mRNA was down-regulated by(83.67±2.56)% .The proliferation of stable-transfected cells was inhibited by(74.33±0.58)% . The adhesion capability of stable-transfected cells was enhanced but the migration capacity was decreased compared with the negative control cells. Conclusion:The pGPU6-Neo-RMP-484 cell lines with stable transfection of RMP siRNA recombinant vector are successfully screened,which can be used as a cellular model for studying the molecular mechanism of RMP. Down-regulation of RMP gene expression can effectively inhibit the proliferation, enhance the adhesion, and decrease the migration of SMMC-7721 cells.
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Objective To investigate the effects of liraglutide on glucose-lipid metabolism in ApoE-/-mice with RNAi-mediated adiponectin gene inhibition. Methods The dose-effective relationship of liraglutide was evaluated by intravenous glucose tolerance test (IVGTT), and the insulin sensitivity and glucose-lipid metabolism were assessed by the hyperinsulinemic-euglycemic clamp technique using 3-[3 H]-glucose as a tracer. Results In the IVGTT, blood glucose was significantly lower in the 1 mg/kg liraglutide group than that in other groups ( all P＜0. 01 ) at the points of 5, 15, and 30 min after glucose load. However, plasma insulin was significantly higher at the points of 5 and 15 min (all P＜0. 01 ). Fasting blood glucose (FBG), body weight, free fatty acids (FFA),total cholesterol, triglycerides, low-density lipoprotein-cholesterol ( LDL-C), and fasting plasma insulin in ApoE-/-mice with co-injection of liraglutide and adiponectin shRNA adenovirus ( HEA group ) were significantly lower than those in ApoE-/-mice with adiponectin shRNA adenovirus injection ( ADI group, P＜0. 05 or P＜0. 01 ). However,high-density lipoprotein-cholesterol (HDL-C) was significantly higher than the latter (P＜0. 05 ). During the steady-state of clamp, plasma insulin in ADI group was significantly higher than that in HEA group (P＜0. 01 ). Although FFA, total cholesterol, and triglycerides were suppressed in all groups, they were still higher in ADI group than those in HEA group (P＜0. 05). Glucose infusion rate (GIR) in HEA group were significantly higher than that in ADI group ( P ＜ 0. 01 ). At the end of clamp, glucose disappearance rate ( GRd ) was significantly lower, and hepatic glucose production significantly higher in ADI group than those in HEA group (P＜0.01 ). Conclusion Administration of liraglutide may ameliorate insulin resistance via increasing plasma adiponectin level in ApoE-/-mice with RNAi-mediated adiponectin gene inhibition.
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Objective To determine the effect of signal transducers and activators of transcription 3 (STAT3) gene silencing by shRNA mediated by lentiviral vector for the treatment of colorectal cancer. Methods The recombinant lentiviral vector pRNAT-shSTAT3, empty lentiviral vector pRNAT-GFP, and lentiviral packaging plasmids in supernatant were collected to transfect HT-29 cells for harvesting the HT-29-shSTAT3 cells and HT29-GFP cells. Fifteen male rats were divided into three groups (n = 5 ), and then they were inoculated with HT-29cells, HT-29-GFP cells and HT-29-shSTAT3 cells, respectively. Cell growth was assessed by MTT assay and the changes in cell cycle were detected by flow cytometry. The changes in microvessel density (MVD) of tumors were detected by immunohistochemistry. All data were analysed by one-way analysis of variance. Results The growth of HT-29-shSTAT3 cells was significantly suppressed compared with HT-29 and HT-29-GFP cells (F = 632.50,P ＜ 0. 05 ). The proportions of cells at the G0/G1 phase were 68.7% ± 2.9% in HT-29-shSTAT3 cells, 38.5% ±1.6% in HT-29-GFP cells and 38.7% ± 2.3% in HT-29 cells, with a significant difference among the three groups (F = 166.53, P ＜ 0.05 ). The MVDs of HT-29 cells, HT-29-GFP cells and HT-29-shSTAT3 cells were 29 ±5, 28 ±4 and 10 ±3, respectively, with a significant difference among the three groups (F=31.60, P ＜0.05). Conclusion STAT3 gene silencing by shRNA mediated by lentiviral vector can significantly inhibit the growth of colorectal cancer cells.
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Objective: To construct a recombinant lentivirus harboring RNAi sequence targeting rat nogo receptor gene and to observe its infection efficiency of 293T cells. Methods: Lentivirus shuttle plasmid containing siNgR199 cDNA was constructed by gene engineering and was used to transfect 293T cells in the presence of packaging plasmids. Forty-eight hours later the supernatant was collected and the titer of virus was determined. The recombinant lentivirus and the standard lentivirus were used to transfect 293T cells at 1 MOI,3 MOI,5 MOI,10 MOI and 20 MOI. Polymerase chain reaction (PCR) was used to detect the recombinant vector; enhanced green fluorescent protein (EGFP) expression was used to determine the titer and the infection rate of the recombinant lentivirus under fluorescent microscope. Results: Restriction endonuclease and PCR analysis confirmed that the siNgR199 cDNA was successfully inserted into the lentivirus vector. The titer of the recombinant lentivirus harboring siNgR199 was 1×108 ifu/ ml and the best MOI was 3. Conclusion: The recombinant lentivirus containing siNgR199 gene has been successfully constructed, which lays a foundation for future axon regeneration in treatment of spinal cord injury.
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Objective To construct the short hairpin RNA recombinant plasmids targeting mdr1 gene which expresses highly in ovarian cancer resistance strain SKOV3/TAXOL to silence endogenefic mdr1 gene expression and investigate the role of mdr1 gene in the development of resistant ovarian cancer. Methods The pGPU6/GFP/Neo-mdr1 were constructed by gene clone technology. The influence on proliferation and apoptosis were investigated by CCK-8 in SKOV3/TAXOL after transfected pGPU6/GFP/Neo-mdr1. Results The expression against mdr1 proteins were inhibited by pGPU6/GFP/Neo-mdr1. The cell proliferation were inhibited after transfected pGPU6/GFP/Neo-mdr1 by CCK-8. The apoptosis were observed in DAB experiments and the apoptosis rate increased. Conclusion mdr1 plays an important role in proliferation of resistant ovarian cancer and the short hairpin RNA of mdr1 can efficiently suppress mdr1 expression and enhance the apoptosis in SKOV3/ATAXOL.
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Objective To determine the effect of 2 transforming growth factor β1 (TGF-β1) short hairpin RNA (shRNA) expression plasmids (pcDU6-A1-A2 and pcDU6-B1-B2) on proliferation, TGF-β1, connective tissue growth factor (CTGF), and fibronectin (FN) expression induced by human serum albumin (HAS) in HK2 cells. Methods A vector plasmid containing the TGF-β1 shRNA was generated. An HK2 cell line was used in the study. The 2 TGF-β1 shRNA expression plasmids were transfected into cultured HK2 cells by lipofectamine 2000. Cellular proliferation was assessed by tetrazolium salt colorimetry. The semi-quantitative reverse transcriptive PCR was performed to detect the expression of TGF-β1,CTGF, and FN mRNA. Levels of TGF-β1 and FN protein were measured with a sandwich enzyme-linked immunosorbent assay. Results After treating with 5 g/L HAS for 24 hours in HK2 cells, cellular proliferating capacity increased significantly (P＜0.05). The expression levels of TGF-β1, CTGF, and FN mRNA were upregulated in HK2 cells stimulated by 5 g/L HAS, and levels of TGF-β1 and FN protein in the culture supernatant increased (P＜0.05). The introduction of pcDU6-A1-A2 and pcDU6-B1-B2 resulted in significant reduction of cellular proliferation activity, and the expression levels of TGF-β1, CTGF, and FN mRNA were downregulated (P＜0.05). Levels of TGF-β1 and FN protein in the culture supernatant decreased (P＜0.05) after 12 or 24 hours of TGF-β1 shRNA transfection into HK2 cells There was no significant difference in the expression levels of TGF-β1, CTGF, and FN mRNA between the 2 pcDU6 vector plasmid mediated TGF-β1 shRNA groups (P＞0.05). Conclusion pcDU6 vector plasmid mediated TGF-β1 shRNAs could obviously inhibit the expression levels of TGF-β1, CTGF, FN and cellular proliferation stimulated by HAS in HK2 cells, which may be related to the mediation of TGF-β1.
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Objective To investigate the influence of lentivirus mediated short hairpin RNA(shRNA)target to human papilloma virus(HPV)16 E6 on invasive ability of cervical cancer Caski cells.Methods Lentivirus was produced after shRNA target to human papilloma virus(HPV)16 E6 and to nonsense was cloned to lentivirus work vector.Infection ratio was assessed by assay of EGFP positive cells of Caski.Total mRNA of E6 was determined by RT-PCR after Caski cells were infected by lentivirus.The change of E6 protein expression was analyzed by Western blot.The invasive ability of Caski cells was assayed employing Transwell.Results The optimal MOI (Multiplicity of infection)of lentivirus to Caski was 2.5.Total mRNA and protein of E6 were decreased (by 70%and 63%)in interfering group compared with control group.The invasive ability of Caski cells also reduced after infected by lentivirus.Conclusion shRNA mediated by lentivirus can inhibit expression of HPV16 E6 and invasive ability of cervical cancer cells.
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Objective To design and construct a vector of signal transducer and activator of transcription 3 (STAT3) small interfering RNA (siRNA),and to investigate the effect of the recombinant plasmid on the proliferation and apoptosis of gastric cancer cell SGC7901. Methods The PAVU6+27-STAT3 siRNA expression vector was constructed, then transfected into the cultured gastric cancer cells SGC7901 by DOTAP method. STAT3 mRNA and protein in SGC7901 were determined by RT-PCR and Western blotting respectively. Cell proliferation was tested by MTT and 3H-TdR incorporation. Cell apoptosis percentage and bcl-2 expression were observed by TUNEL and flow cytometry respectively. Results The PAVU6+27-STAT3 siRNA expression vector was successfully constructed. In the transfected SGC7901 cells, cell proliferation, 3H-TdR incorporation and bcl-2 expression were decreased, and cell apoptosis percentage was increased. Conclusion PAVU6+27-STAT3 siRNA expression vector may be an efficient method to inhibit the proliferation and promote the apoptosis of gastric cancer cells.
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Objective:To clone the recombinant plasmid affecting gene DNMT1 expression by RNA interfering and transfect it into AGS in order to further search its effects on the cell cycle of gastric cancer AGS.Methods:Two DNA sequences containing short hairpin structure was designed and synthesized.The complement form was obtained by annealing and inserted into vector pTZU6+1.The recombinant plasmid was transfected into AGS.Finally the cell cycle was evaluated through flow cytometer.Results:The S cycle cells of the transfected AGS dropped dramatically.And the G2-M cycle cells increased significantly.Conclusion:Successful cloning and transfecting the recombinant make it possible to search new gene therapy for the tumors.
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Objective:To investigate the proliferating inhibition and the mechanisms of dsRNA interference c-erbB-2 and c-raf-1 genes expression combined transfection in the human tongue carcinoma Tca8113 cell lines.Methods:There were 3 groups in our study,control group,normal control group and RNAi experimental group.At different time after liposome-mediated transfection,the cell proliferation,apoptosis,mRNA level,protein expressing level and cell cycle were observed by MTT,RT-PCR and electronic microscope.Results:According to the results of RNAi experimental group,the OD-value were 0.073( P